1. Field of the Invention
The present invention relates to assays that detect and characterize single or linked mutations in a genome of a Hepatitis C Virus (HCV) that are associated with resistance of a subject to an anti-HCV drug. The assays can also be used for predicting resistance to an anti-HCV drug of a subject infected with HCV prior to or early during antiviral therapy or for selecting an alternative therapy for an HCV-infected subject that has developed resistance to a particular therapeutic drug or drug combination. The invention also relates to nucleotide primer pairs and kits for carrying out these assays.
2. Description of Related Art
HCV was cloned and characterized about 15 years ago by Choo and colleagues. Choo et al. (1989) Science 244, 359-362. HCV belongs to the family of Flaviviridae and comprises an enveloped nucleocapsid and a single-stranded RNA genome of positive polarity. (Bartenschlager et al. (2003) Antiviral Res 60, 91-102.) The HCV genome consists of 5′ and 3′ noncoding (UTR or NCR) regions that flank a single long open reading frame (ORF). This ORF encodes three structural proteins at the amino-terminal end and six nonstructural (NS) proteins at the carboxy-terminal end. The structural proteins are the nucleocapsid core protein (C) and the two glycoproteins envelope 1 (E1) and envelope 2 (E2). The non-structural proteins are named NS2, NS3, NS4a, NS4b, NS5a, NS5b. The 5′NCR is the most highly conserved region of the HCV genome, whereas the sequences of the two envelope proteins (E1 and E2) are highly variable among different HCV isolates. The highest degree of variation has been observed in a region within E2, now commonly termed hypervariable region 1.
Since the initial identification of HCV, at least 7 different major viral types have been identified and designated genotype 1 through 7. Within these genotypes are numerous subtypes (e.g. HCV1a, 1b, 1c). Genotype and subtype of a virus with which a subject is infected may affect clinical prognosis as well as responsiveness to various drug treatments. (Simmonds et al. (1995) Hepatology 21, 570-582; Bukh et al. (1995) Semin Liver Dis 15, 41-63; Chevaliez and Pawlotsky (2007) World J Gastroenterol 13, 2461-2466).
HCV infection remains a serious medical problem to this date. There are currently about 170 million people infected with HCV. HCV is transmitted primarily by blood and blood products as well as by vertical transmission during pregnancy. The initial course of infection is typically mild. However, the immune system is often incapable of clearing the virus, and subjects with persistent infection are at a high risk for liver cirrhosis and hepatocellular carcinoma. (Poynard et al. (1997) Lancet 349, 825-832).
Current standard treatment for chronic HCV infection is based on a combination of pegylated interferon alpha and ribavirin. This therapy produces a sustained anti-viral response in 85-90% of subjects infected with genotypes 2 and 3, but, unfortunately, only in about 45% of subjects infected with the prevalent genotype 1. (Stribling et al. (2006) Gastroenterol Clin North Am vol, 463-486.) Additional therapies using other drugs and drug combinations that are endowed with higher antiviral activity and superior safety profiles are clearly required, in particular for the prevention of HCV recurrence.
Introduction of diagnostic tests for screening blood products has significantly reduced the rate of new infection. Availability of in vitro models, i.e., HCV subgenomic replicon models and an infectious cell culture model, and improvements in molecular research techniques such as the Polymerase Chain Reaction (PCR) have facilitated development of additional potent inhibitors of HCV replication targeting directly a viral protein or acting indirectly through host proteins involved in viral infection. (Bartenschlager (2002) Nat Rev Drug Discov 1, 911-916; Wakita et al. (2005) Nat Med 11, 791-796.) Several of these new compounds have entered clinical trials or are already on the market (http://www.hcvadvocate.org/hepatitis/hepC/HCVDrugs—2007.pdf).
Assays have been developed that are aimed at providing prognostic information about the likelihood of responsiveness to an anti-HCV therapy. (Gretch et al. (1997) Hepatology 26, 43s-47s; Podzorski (2002) Arch Pathol Lab Med 126, 285-290). These assays include serological tests and qualitative or quantitative molecular tests. Examples of PCR-based assays of HCV viral load are Cobas Amplicor® (Roche) and m2000 Real-Time PCR Diagnostics System® (Abott). Other PCR-based assays that include, e.g., Versant® HCV Genotyping Assay (Bayer Diagnostics), INNO-LiPA HCV II® (Innogenetics), GEN-ETI-K DEIA kit (Sorin, Saluggia, Italy) and TRUGENE HCV 5′NC genotyping kit (Visible Genetics Europe, Evry, France) identify HCV genotype and subtype. Systematic assessment of HCV genotype prior to therapy has been advocated recently because HCV genotype will determine choice and dose regimen of the most effective anti-HCV drug, e.g. ribavirin or interferon, as well as duration of treatment. Current genotype identification relies primarily on sequencing of a small subregion of an HCV genome, e.g., the 5′UTR, but not of a full or nearly full HCV genome.